ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2018, Vol. 44, No. 1, pp. 90–103. © Pleiades Publishing, Ltd., 2018. Original Russian Text © D.D. Zhdanov, Y.A. Gladilina, D.V. Grishin, V.S. Pokrovsky, M.V. Pokrovskaya, S.S. Aleksandrova, N.N. Sokolov, 2018, published in Bioorganicheskaya Khimiya, 2018, Vol. 44, No. 1, pp. 87–101.
Apoptotic Endonuclease EndoG Induces Alternative Splicing of Telomerase TERT Catalytic Subunit, Caspase-2, DNase I, and BCL-x in Human, Murine, and Rat CD4+ Т Lymphocytes D. D. Zhdanova, 1, Y. A. Gladilinaa, D. V. Grishina, V. S. Pokrovskya, b, M. V. Pokrovskayaa, S. S. Aleksandrovaa, and N. N. Sokolova aOrekhovich
Institute of Biomedical Chemistry, Moscow, 119121 Russia Blokhin Cancer Research Center, Moscow, 115478 Russia
b
Received May 15, 2017; in final form, June 9, 2017
Abstract⎯Apoptotic endonuclease EndoG plays a key role in the alternative splicing of mRNA of human TERT telomerase catalytic subunit. The aim of this work was to test the ability of EndoG to induce alternative splicing of mRNA of other genes and in other organisms. To determine new mRNA splice-variants, EndoG overexpression was induced in human, mouse and rat CD4+-T-lymphocytes followed by sequencing of total RNA of these cells. Sequencing results showed that besides TERT, EndoG induced alternative splicing of deoxyribonuclease I (DNase I), caspase-2 (Casp-2) and BCL-x. The expression level of EndoG strongly correlated with mRNA splicing-variants of TERT, DNase I, Casp-2, and BCL-x in intact CD4+-T cells of healthy donors as well as different lines of mice and rats. EndoG overexpression induced down-regulation of fulllength mRNAs of TERT, DNase I, Casp-2, and BCL-x and up-regulation of their short-length mRNAs. Alternative splicing of studied mRNAs resulted in down-regulation of enzymatic activity of proteins in vitro and in vivo. The results of this work confirm the ability of endonuclease EndoG to induce alternative splicing of several mRNAs in human, mice and rats. Keywords: alternative splicing, EndoG, TERT, Casp-2, DNase I, BCL-x DOI: 10.1134/S1068162018010181
the AS mechanism similar to splicing switching oligonucleotides [7, 8]. Induced by EndoG, AS of hTERT mRNA resulted in inhibition of telomerase activity in cells, telomere shortening to critical values and apoptosis of human cells [6]. It is of interest to study the ability of EndoG to induce AS of mRNA of other genes. In this paper, we investigated the effect of EndoG on the AS of some genes and on the catalytic activity of proteins encoded by them in activated human, murine and rat CD4+-T cells.
Alternative splicing (AS) of mRNA is one of the ways to regulate the catalytic activity and function of proteins [1]. The main mechanisms of AS are the exposure of the cis element, the selection of the splice site mediated by RNA-dependent adenosine deaminase, the convergence of the cis elements, the twisting of the introns, as well as the competitive phenomena in the secondary structure of the RNA [2, 3]. A number of proteins participating in these processes have been identified [4]. However, how the AS works is not fully understood. In previous studies, we showed that the apoptotic endonuclease EndoG induces AS of mRNA of the catalytic subunit of telomerase hTERT (human Telomerase Reverse Transcriptase) [5, 6]. Increased expression of EndoG, for example in response to DNA damage, results in the formation of a small EGPO (EndoG-produced oligonucleotide) RNA that causes
RESULTS AND DISCUSSION Results of RNA sequencing. To determine the mRNA of the genes in the splicing of which EndoG participates, we induced the expression of EndoG by transfection of human, mouse and rat CD4+-T-lymphocytes with the pEndoG-GFP plasmid. Control transfection was performed with the plasmid pGFP encoding the GFP fluorescent protein. Induction of EndoG expression was also performed by incubating cells with a DNA damaging agent, cisplatin. The total RNA of cells was sequenced. Analysis of mRNA levels of splice variants of various genes has shown that both in cells transfected with pEndoG-GFP and in cells
1 Corresponding
author: phone: +7 (910) 478-12-75; fax: +7 (499) 245-08-57; e-mail:
[email protected]. Abbreviations: AS, alternative splicing; TERT, telomerase reverse transcriptase; EndoG, endonuclease G; EGPO, EndoG-produced oligonucleotide; GAPDH, glyceraldehyde-3phosphatedehydrogenase; GFP, green fluorescent protein; TRAP, telomeric repeats amplification protocol.
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incubated with cisplatin, the number of mRNA splice variants of the four following genes changes: the catalytic subunit of telomerase (TERT), caspase-2 (Casp-2), deoxyribonuclease I (DNase I) and B-cell lymphomaextra (BCL-x) (Table 1). Overexpression of EndoG caused a decrease in the amount of mRNA of the fullsize (α+β+) variant of TERT and an increase in the (α+β–)-splice variant of TERT, which is formed upon deletion of exons 7 and 8 of pre-mRNA TERT [9]. At the same time, a decrease in the level of the mRNA of the full-length version of Casp-2L and an increase in the mRNA of the truncated splice version of Casp-2S were detected. The latter is formed as a result of the appearance of the stop-codon due to the shift of the reading frame when the additional coding exon 9 is activated [10]. An increase in EndoG expression was accompanied by a decrease in the amount of fulllength DNase I mRNA and an increase in the level of Δ4DNase I mRNA resulting from the deletion of exon 4. This resulted in a decrease in the number of full-length BCL-xL mRNA and an increase in the BCL-xS mRNA level resulting from the deletion of 189 nucleotides in exon 2. The change in the proportion of the mRNA of splice variants of the studied genes was observed both in human cells and in mouse and rat cells. The revealed shift in the ratio of splice variants of the mRNA of these genes was not accompanied by a change in the total amounts of their mRNA. Transfection of cells with the control plasmid pGFP did not result in a change in the proportion of the mRNA of the studied genes. EndoG expression correlates with the expression of splice variants of TERT, Casp-2, DNase I, and BCL-x. To study the relationship between the level of expression of EndoG and the number of splice variants of TERT, Casp-2, DNase I, and BCL-x, the mRNA expression of these genes in human, murine and rat CD4+-T lymphocytes was estimated by real-time RT-PCR. The results showed a wide range of expression levels for both EndoG and splice variants of the studied genes (Fig. 1) in healthy donors, as well as in various lines of mice and rats. When ranking the cells according to the level of relative expression of EndoG, the median was 0.299 for human CD4+-T cells and 0.311 and 0.329 for mouse and rat cells of different lines, respectively. The correlation analysis showed the relationship between the expression levels of EndoG and the splice variants of TERT, Casp-2, DNase I, and BCL-x (Fig. 2). In cells with a relatively low expression of EndoG (below the median), following full-size splice variants (α+β+)hTERT, Casp-2L, DNase I, and BCL-xL predominate over other splice variants. In groups of cells with a relatively high expression of EndoG (above the median), an increase in the mRNA of short splice variants was revealed: (α+β–)-hTERT, Casp-2S, Δ4DNase I and BCL-xS, and a decrease in mRNA level of full-length variants. The correlation coefficients of EndoG expresRUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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sion with splice variants of the studied genes are presented in Table 2. For further work, CD4+-T cells of donors 2, 3, 6, and 9, C57BL/6 mice and Wistar rats were chosen, since the correlation between the expression of EndoG and the mRNA of the splice variants of the studied genes in these samples was most pronounced. Overexpression of EndoG induces AS of TERT, Casp-2, DNase I, and BCL-x. To confirm the role of EndoG in the process of the AS of mRNA of TERT, Casp-2, DNase I, and BCL-x, the expression of its gene was induced by a DNA damaging agent cisplatin at a concentration of 60 μM [11], also, the cells were transfected with plasmid pEndoG-GFP or control plasmid pGFP. The applied concentration of cisplatin was selected by us earlier during preliminary experiments (data not shown). Using the TUNEL method for flow cytometry, we observed how the number of cells with damaged DNA changes after incubation with cisplatin. Incubation with cisplatin caused an increase in DNA damage in human CD4+-T cells, as well as in mice and rats (Figs. 3a–3d). Incubation with cisplatin, as well as transfection with plasmid pEndoG-GFP, resulted in a significant increase in the expression of EndoG (Figs. 3e–3k) in human, mouse and rat cells. A significant increase in EndoG mRNA was confirmed by real time RT-PCR (Figs. 3e–3g), as well as by Western blotting (Figs. 3h–3k). Overexpression of EndoG was accompanied by a change in the mRNA pool of the TERT, Casp-2, DNase I, and BCL-x splice variants: an increase in the mRNA of truncated splice variants ((α+β–)-hTERT, Casp-2S, Δ4DNase I and BCL-xS) and a decrease in the number of mRNAs of full-length variants (α+β+)-hTERT, Casp-2L, DNase I, and BCL-xL) in human (Figs. 4a–4d), murine (Figs. 4e–4h) and rats (Figs. 4i–4l) cells. EndoG causes a decrease in the pool of full-length forms of TERT, Casp-2, DNase I, and BCL-x and a decrease in their enzymatic activity in vitro. We attempted to elucidate the effect of induction of truncated splice variants of mRNA encoding TERT, Casp-2, DNase I and BCL-x, on the enzymatic activity of these proteins in cells. Results of Western blotting using antibodies to the studied proteins showed that transfection of cells with pEndoG-GFP, as well as their incubation with cisplatin, resulted in a significant decrease in the number of full-length forms of proteins (α+β+)-hTERT, Casp-2L, DNase I and BCL-xL and an increase in the number of truncated forms (α+β–)hTERT, Casp-2S, Δ4DNase I, and BCL-xS in human, murine and rat CD4+-T lymphocytes (Figs. 5a–5j). By Western blotting, the change (decrease) in the amount of only the full-length protein form of DNase I was detected, since the Δ4DNase I form was not detected by the antibodies used. To analyze the activity of telomerase in cell extracts after transfection or incubation of cells with cisplatin, Vol. 44
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Table 1. The level of mRNA of investigated genes splice variants relatively to control cells mRNA splice variants
pEndoG-GFP
pGFP
Cisplatin
Human Total TERT
1.112
0.912
1.206
(α+β+)-TERT
0.534 (*, #)
1.087
0.604 (*)
(α+β–)-TERT
2.755 (*, #)
1.101
1.959 (*)
Total Casp-2
0.088
0.833
1.099
Casp-2L
0.457 (*, #)
1.105
0.360 (*)
Casp-2S
1.967 (*, #)
0.950
1.773 (*)
Total DNase I
1.233
1.118
0.968
DNase I
0.324 (*, #)
0.867
0.504 (*)
Δ4DNase I
2.967 (*, #)
0.997
2.032 (*)
Total BCL-x
1.300
1.081
1.166
BCL-xL
3.066 (*, #)
0.937
2.241 (*)
BCL-xS
0.395 (*, #)
0.881
0.541 (*)
Mouse Total TERT
0.917
0.820
0.993
(α+β+)-TERT
0.636 (*, #)
0.112
0.521 (*)
(α+β–)-TERT
2.299 (*, #)
1.130
2.002 (*)
Total Casp-2
1.135
1.056
0.067
Casp-2L
0.317 (*, #)
1.138
1.244 (*)
Casp-2S
1.866 (*, #)
1.002
0.866 (*)
Total DNase I
0.806
1.244
0.964
DNase I
0.355 (*, #)
0.951
0.637 (*)
Δ4DNase I
2.957 (*, #)
0.997
2.250 (*)
Total BCL-x
0.964
1.051
1.096
BCL-xL
0.235 (*, #)
1.170
0.355 (*)
BCL-xS
3.677 (*, #)
1.167
3.051 (*)
Rat Total TERT
1.061
1.120
1.008
(α+β+)-TERT
0.196 (*, #)
0.961
0.412 (*)
(α+β–)-TERT
4.677 (*, #)
0.883
2.352 (*)
Total Casp-2
0.930
0.900
0.794
Casp-2L
0.561 (*, #)
1.062
0.520 (*)
Casp-2S
2.347 (*, #)
0.875
2.214 m
Total DNase I
0.819
1.029
0.907
DNase I
0.404 (*, #)
0.866
0.631 (*)
Δ4DNase I
2.031 (*, #)
0.974
2.000 (*)
Total BCL-x
1.007
1.153
1.095
BCL-xL
0.388 (*, #)
1.068
0.304 (*)
BCL-xS
1.905(*, #)
1.251
1.855 (*)
Expression levels with p < 0.05 were considered statistically significant. * p < 0.05 relatively to the intact control cells; #p < 0.05 relatively to the cells transfected with pGFP RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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Normalized expression level, conventional units
Normalized expression level, conventional units
Normalized expression level, conventional units
No. 1
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0
0.1
0.2
0.3
0.4
0.5
0.6
EndoG
1 2 3 4 5 6 7 8 910 1112 Donor
SD
(k)
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
(f)
0
0.2
0.4
0.6
0.8
WKY DA Wistar SHR Rat strain
EndoG
Mouse strain
ICR BALB/c C57BL/10 C57BL/6 DBA/2 SJL C3H CBA
Normalized expression level ×10е–3, conventional units Normalized expression level ×10е–3, conventional units Normalized expression level ×10е–3, conventional units
(l) 16 14 12 10 8 6 4 2 0 SD WKY DA Wistar SHR Rat strain (α+β+)-TERT (α+β–)-TERT
TERT
Mouse strain (α+β+)-TERT (α+β–)-TERT
1 2 3 4 5 6 7 8 9 101112 Donor (α+β+)-TERT (α+β–)-TERT (g) TERT 20 18 16 14 12 10 8 6 4 2 0
TERT
ICR BALB/c C57BL/10 C57BL/6 DBA/2 SJL C3H CBA
(b) 18 16 14 12 10 8 6 4 2 0
(c) Casp-2
0
0.1
0.2
0.3
0.4
0.5
0.6
(d) DNase I
SD
WKY DA Wistar SHR Rat strain Casp-2L Casp-2S
Casp-2
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 SD
(n)
WKY DA Wistar SHR Rat strain DNase I Δ4DNase I
DNase I
Mouse strain DNase I Δ4DNase I
1 2 3 4 5 6 7 8 9 101112 Donor DNase I Δ4DNase I (i) DNase I 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Mouse strain Casp-2L Casp-2S (m)
0
0.05
0.10
0.15
1 2 3 4 5 6 7 8 9 101112 Donor Casp-2L Casp-2S (h) Casp-2 0.20
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Normalized expression level, conventional units Normalized expression level, conventional units
EndoG
ICR BALB/c C57BL/10 C57BL/6 DBA/2 SJL C3H CBA
Normalized expression level, conventional units Normalized expression level, conventional units Normalized expression level, conventional units
Normalized expression level, conventional units Normalized expression level, conventional units Normalized expression level, conventional units
ICR BALB/c C57BL/10 C57BL/6 DBA/2 SJL C3H CBA
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(a)
(e) BCL-x
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 SD
(o)
0
0.1
0.2
0.3
0.4
1 2 3 4 5 6 7 8 9 101112 Donor BCL-xL BCL-xS (j) BCL-x 0.5
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
WKY DA Wistar SHR Rat strain BCL-xL BCL-xS
BCL-x
Mouse strain BCL-xL BCL-xS
ICR BALB/c C57BL/10 C57BL/6 DBA/2 SJL C3H CBA
1.0
APOPTOTIC ENDONUCLEASE EndoG INDUCES ALTERNATIVE SPLICING 93
Fig. 1. Level of expression of EndoG and splice variants of TERT, Casp-2, DNase I, and BCL-x in human, murine, and rat CD4+-T lymphocytes. EndoG expression level (a, f, k) and splice variants of TERT (b, g, l), Casp-2 (c, h, m), DNase I (d, i, n), and BCL-x (e, j, o) in CD4+-Т cells of 12 healthy donors (a–e), mice of various strains (f–j), rats of various strains (k–o) were shown. Gene expression levels were measured by real time RT-PCR and normalized using the expression level of the 18S rRNA reference gene.
ZHDANOV et al.
0.6
*
1.0 0.8 0.6 0.4 0.2 0
High Low level of level of EndoG EndoG
0.5 0.4 0.3 0.2 0.1 0
High Low level of level of EndoG EndoG
High Low level of level of EndoG EndoG
(α+β+)-TERT (α+β−)-TERT *
0.5 0.4 0.3 0.2 0.1 0
Low High level of level of EndoG EndoG
16 14 12 10 8 6 4 2 0
TERT *
(h) 0.12
0.6
0.10 0.08 0.06 0.04 0.02 0
High Low level of level of EndoG EndoG
0.5 0.4 0.3 0.2 0.1 0
High Low level of level of EndoG EndoG
14 12 10 8 6 4 2 0
(m)
High Low level of level of EndoG EndoG
Casp-2 * *
0.5
*
0.4 0.3 0.2 0.1 0
High Low level of level of EndoG EndoG
(α+β+)-TERT (α+β−)-TERT
Casp-2L Casp-2S
DNase I Δ4DNase I DNase I * *
0.2 0.1
BCL-x * *
0.5 0.4 0.3 0.2 0.1 0
High Low level of level of EndoG EndoG
0.3
0
High Low level of level of EndoG EndoG BCL-xL BCL-xS BCL-x
(j)
*
0.4 0.3 0.2 0.1 0
High Low level of level of EndoG EndoG
High Low level of level of EndoG EndoG
DNase I Δ4DNase I (n)
0.5
*
0.6 0.5 0.4 0.3 0.2 0.1 0
BCL-xL BCL-xS (o)
DNase I
0.7 Expression level, conventional units
*
TERT
0.6
0.4
Casp-2L Casp-2S
Expression level, conventional units
Expression level, conventional units
0.6
(l) Expression level ×10е–3, conventional units
EndoG
(e)
0.5
High Low level of level of EndoG EndoG
(α+β+)-TERT (α+β−)-TERT (k)
(i)
Expression level, conventional units
Expression level, conventional units
0.6
(g)
Expression level, conventional units
EndoG
Expression level ×10е–3, conventional units
(f)
Casp-2L Casp-2S Casp-2 *
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
DNase I * *
Expression level, conventional units
1.2
(d)
Casp-2 * *
Expression level, conventional units
*
(c)
TERT *
Expression level, conventional units
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
(b)
Expression level, conventional units
EndoG
Expression level ×10е–3, conventional units
Expression level, conventional units
(a)
Expression level, conventional units
94
BCL-x *
0.4 0.3 0.2
High Low level of level of EndoG EndoG DNase I Δ4DNase I
0.1 0
High Low level of level of EndoG EndoG BCL-xL BCL-xS
Fig. 2. Expression level of splice variants of TERT (b, g, l), Casp-2 (c, h, m), DNase I (d, i, n), and BCL-x (e, j, o) in cases of high and low EndoG expression level (a, f, k) in CD4+-Т cells of healthy donors (a–e), mice of various strains (f–j), rats of various strains (k–o). Gene expression levels were measured by real time RT-PCR and normalized using the expression level of the 18S rRNA reference gene. * p ≤ 0.05 for the genes’ expression levels between cell groups.
the telomeric repeat amplification protocol (TRAP) was used. In transfected pEndoG-GFP or human, murine and rat cells incubated with cisplatin, a significant decrease in telomerase activity was observed to
10–50% of the control cell level (Figs. 5k–5l). Overexpression of EndoG was accompanied by a 2.5- to 3-fold decrease in Casp-2 activity in lysates of transfected pEndoG-GFP or in human, murine and rat cells
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Table 2. Coefficient of correlation of the expression of EndoG and splice variants of TERT, Casp-2, DNase I, and BCL-x genes in human, murine and rat CD4+-T lymphocytes Human CD4+-Т lymphocytes mRNA splice variant
(α+β+)-hTERT (α+β–)-hTERT Casp-2L Casp-2S DNase I Δ4DNase I BCL-xL BCL-xS
Murine CD4+-Т lymphocytes
Rat CD4+-Т lymphocytes
high EndoG low EndoG low EndoG high EndoG low EndoG high EndoG expression level expression level expression level expression level expression level expression level (N = 6) (N = 6) (N = 4) (N = 4) (N = 3#) (N = 2#) 0.751 (*) 0.808 (*) 0.701 (*) 0.612 (*) 0.615 0.833 (*) 0.702 (*) 0.602
0.841 (*) 0.764 (*) 0.722 (*) 0.608 (*) 0.706 0.777 (*) 0.810 (*) 0.700 (*)
0.625(*) 0.751 0.734 (*) 0.729 (*) 0.688 (*) 0.724 0.641 (*) 0.680 (*)
0.731 (*) 0.691 (*) 0.738 (*) 0.725 (*) 0.721 (*) 0.680 (*) 0.782 0.696 (*)
0.523 0.344 0.537 0.612 0.468 0.312 0.516 0.445
0.680 0.482 0.396 0.604 0.390 0.591 0.358 0.427
# Correlation coefficients are not statistically reliable due to the small sample size; * correlation coefficients are statistically significant, p ≤ 0.01 using Pearson’s correlation criterion.
incubated with cisplatin (Fig. 5m). The study of DNase I activity by the method of zymography showed a 2.5- to 4-fold decrease in the activity of this enzyme in cells with EndoG overexpression (Figs. 5n, 5o). We failed to assess the effect of the change in the ratio of the forms of BCL-x on its enzymatic activity in cells, since there is no test system for measuring the activity of this protein. Induced expression of EndoG causes AS of TERT, Casp-2, DNase I, and BCL-x and a decrease in the enzymatic activity of the corresponding proteins in vivo. To study the ability of EndoG to induce AS of TERT, Casp-2, DNase I and BCL-x in vivo, C57BL/6 mice and Wistar rats were intravenously injected with cisplatin. After 72 hours, CD4+-T lymphocytes were isolated from the blood of the animals and gene expression studies were performed. The TUNEL method for flow cytometry revealed an increase in the number of CD4+-T cells with damaged DNA in the blood of mice and rats after cisplatin administration (Figs. 6a–6c). An increase in the number of cells with damaged DNA was accompanied by an increase in the expression of EndoG in them and an increase in the amount of the corresponding protein, as demonstrated by real-time RT-PCR (Fig. 6d) and confirmed by Western blotting (Figs. 6e–6g). Western blotting results for TERT, Casp-2, DNase I and BCL-x proteins showed that the induction of EndoG expression by cisplatin was accompanied by a significant decrease in the number of full-length forms of proteins (α+β+)-hTERT, Casp-2L, DNase I, and BCL-xL and an increase in the number of truncated forms (α+β–)-hTERT, Casp-2S, Δ4DNase I, and BCL-xS in CD4+ T lymphocytes in mice and rats (Figs. 7a–7j). The change in the pool of studied forms of proteins was accompanied by a decrease in their enzymatic activity. There was a significant decrease in RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
telomerase activity (Figs. 7k and 7l), Casp-2 (Fig. 7m) and DNase I (Figs. 7n and 7o) in CD4+-T lymphocytes of mice and rats after cisplatin administration. The involvement of EndoG apoptotic endonuclease in the process of AS of hTERT mRNA was demonstrated earlier in human CaCo-2 intestinal carcinoma cells [6] and activated human T lymphocytes [12]. The question of the ability of EndoG to induce AS of other genes in other organisms is currently relevant. To answer this question, we induced the expression of EndoG in human, murine and rat CD4+-T lymphocytes by two methods: we transfected cells with plasmid pEndoG-GFP or incubated them with DNAdamaging agent cisplatin [11]. RNA sequencing showed that the increased expression of EndoG is accompanied by a change in the amount of mRNA of the splice variants of TERT, Casp-2, DNase I, and BCL-x in human, murine and rat CD4+-T cells (Table 1). Two variants account for most of the total human TERT mRNA. Deletion of 36 nucleotides in exon 6 (α-variant) causes the removal of a portion of the hTERT reverse transcriptase domain A and leads to a loss of catalytic activity. The deletion of 182 nucleotides of exons 7 and 8 (the β-variant) results in a shift of the reading frame and the appearance of a stop codon in exon 10, which causes the synthesis of a truncated variant of hTERT [9, 13] functioning as a dominant negative one [14]. In this paper, we showed that EndoG cause an increase in the mRNA (α+β–)splice variant of TERT not only in human but also in murine and rat cells. Previously, it was found that DNA damage by various agents, oxidative stress, and heat shock cause changes in the proportion of Casp-2 splice variants in cells [15–19]. These data are consistent with the Vol. 44
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Cell number
100 100 80 86.35% 80 5.88% 60 60 40 40 20 20 0 0 0 1 1e 1e 1e3 1e0 1e1 1e3 –1 2 1e 1e 1e–1 1e2 TUNEL
Cisplatin
Control
100 100 80 80 88.98% 11.84% 60 60 40 40 20 20 0 0 0 1 1e 1e 1e3 1e0 1e1 1e3 –1 2 1e 1e 1e–1 1e2 TUNEL
(e)
Cisplatin
100 100 80 80 84.64% 10.83% 60 60 40 40 20 20 0 0 0 1 1e 1e 1e3 1e0 1e1 1e3 –1 2 1e 1e 1e–1 1e2 TUNEL
Normalized expression level, conventional units
Normalized expression level, conventional units
Mouse *# 1.4 1.2 1.0 * 0.8 0.6 0.4 0.2 0 pEndoG-GFP Cisplatin pGFP Control (j)
(i)
(h) Human EndoG GAPDH (32 kDa) (36 kDa)
pEndoG-GFP
pGFP
*
60 40 20 0 Human Mouse Rat Cisplatin
Control
Rat 1.2 *# 1.0 0.8 * 0.6 0.4 0.2 0 pEndoG-GFP Cisplatin pGFP Control
(k) Rat EndoG GAPDH (33 kDa) (36 kDa)
Mouse EndoG GAPDH (33 kDa) (36 kDa)
pEndoG-GFP
*
*
80
(g)
(f) Human *# 2.0 1.8 1.6 1.4 1.2 * 1.0 0.8 0.6 0.4 0.2 0 pEndoG-GFP Cisplatin pGFP Control
Control
% TUNEL
Control
(d) 100
Rat
pEndoG-GFP
pGFP
pGFP
Cisplatin
Cisplatin
Cisplatin
Control
Control
Control
2.0 *#
Expression relatively to GAPDH
Cisplatin
(c)
Mouse
Normalized expression level, conventional units
(b)
Human
Cell number
(a)
Cell number
96
*#
*#
1.5 1.0
*
* *
0.5 0 Human Mouse pEndoG-GFP Cisplatin
Rat pGFP Control
Fig. 3. Induction of DNA damage and EndoG expression in cisplatin treated CD4+ cells. The number of human, murine and rat lymphocytes with damaged DNA (a, b and c, respectively) measured by the TUNEL method for flow cytometry; increase in the number of TUNEL-positive cells upon incubation with cisplatin (d) and EndoG expression in human, murine and rat CD4+-T lymphocytes (e, f, and g, respectively) when cultured with cisplatin were shown. Gene expression levels were measured by real time RT-PCR and normalized using the expression level of the 18S rRNA reference gene. Western blotting of EndoG and the reference protein GAPDH in human, murine, and rat cells (h, i, j, respectively) cultured with cisplatin and results of quantification of EndoG against GAPDH (k) were shown. N = 4. *p ≤ 0.05 when compared with control cells; #p ≤ 0.05 when compared with cells transfected with pGFP.
results of our experiments in which cisplatin caused DNA damage and induction of expression of EndoG. There are two splice variants of Casp-2: Casp-2L (Long Form) and Casp-2S (Short Form). The fullsized Casp-2L (435 amino acids), also known as ICH-1 (Interleukin-1β-converting enzyme) consists of the p19 and p12 subunits required for activation and catalytic activity of the enzyme [20]. The splice variant Casp-2S contains an alternative noncoding exon at the 5' end of the mRNA that leads to initiation of translation from the coding codon 2 and the loss of part (31 amino acids) of the CARD domain (Caspase Recruitment Domain) at the N-terminus of the protein [10]. The appearance of an additional coding exon 9 causes a reading frame shift, the appearance of a stop codon at the junction of exons 9 and 10, and a shortening of the C-terminal region of the protein by 92 amino acids [21]. As a result of the AS, Casp-2S
loses the p12 sequence and enzymatic activity. Thus, splice variants of Casp-2 have the opposite effect: Casp-2L induces cell death, while Casp-2S suppresses apoptotic processes [22]. During the development of apoptotic processes EndoG functions simultaneously with another apoptotic endonuclease DNase I [23]. DNase I is found in the cells in the largest amounts [24], is the most active apoptotic DNase, and participates in cisplatininduced activation of the EndoG gene [25]. Induction of AS of DNase I by endonuclease EndoG is accompanied by a decrease in the expression of the full-length variant of DNase I in normal NRK-52E rat kidney cells transfected with plasmid pEndoG-ECFP [26]. BCL-x is a transmembrane mitochondrial protein that regulates the secretion of the contents of mitochondria, for example, cytochromes, into the cytoplasm in the development of apoptotic processes [27,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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No. 1
*#
*
*
(e)
*#
*#
*
TERT *
2018
(i)
*#
*#
*
TERT *
(α+β–)-TERT
(α+β+)-TERT
0 pEndoG-GFP Cisplatin pGFP Control
2
4
6
8
10
12
14
16
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(α+β+)-TERT
0 pEndoG-GFP Cisplatin pGFP Control
1
2
3
4
5
6
(α+β–)-TERT
(α+β+)-TERT
0 pEndoG-GFP Cisplatin pGFP Control
2
4
6
8
10
12
*#
*#
*#
* *
Casp-2
(f) *#
*
Casp-2
(j)
*#
*#
Casp-2
*
*
Casp-2L Casp-2S
0 pEndoG-GFP Cisplatin pGFP Control
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Casp-2L Casp-2S
0 pEndoG-GFP Cisplatin pGFP Control
0.04 *# 0.002
0.06
0.08
0.10
0.12
0.14
0.16
Casp-2L Casp-2S
0 Cisplatin pEndoG-GFP pGFP Control
0.1
0.2
0.3
0.4
0.5
0.6
0.7
(b) 0.8 Normalized expression level, conventional units Normalized expression level, conventional units
TERT
*#
*
DNase I *
*#
DNase I Δ4DNase I
0.9 DNase I * 0.8 0.7 0.6 0.5 * 0.4 *# 0.3 0.2 0.1 0 pEndoG-GFP Cisplatin pGFP Control
(k)
DNase I Δ4DNase I
0 pEndoG-GFP Cisplatin pGFP Control
0.1
0.2
0.3 *#
0.4
0.5
0.6
0.7
(g)
DNase I Δ4DNase I
(c) DNase I 1.8 *# 1.6 * 1.4 1.2 1.0 0.8 0.6 * *# 0.4 0.2 0 pEndoG-GFP Cisplatin pGFP Control Normalized expression level, conventional units Normalized expression level, conventional units
Normalized expression level ×10е–3, conventional units
Normalized expression level ×10е–3, conventional units
Normalized expression level ×10е–3, conventional units
Normalized expression level, conventional units Normalized expression level, conventional units Normalized expression level, conventional units
Vol. 44 Normalized expression level, conventional units
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Normalized expression level, conventional units
(a) 14
*#
*#
*
BCL-x *
*#
*#
*
BCL-x *
(l)
BCL-xL BCL-xS
0.9 BCL-x * *# 0.8 0.7 0.6 0.5 0.4 *# 0.3 0.2 0.1 0 pEndoG-GFP Cisplatin pGFP Control
BCL-xL BCL-xS
0 pEndoG-GFP Cisplatin pGFP Control
0.1
0.2
0.3
0.4
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0.6
0.7
(h)
BCL-xL BCL-xS
0 pEndoG-GFP Cisplatin pGFP Control
0.05
0.10
0.15
0.20
0.25
(d) 0.30
APOPTOTIC ENDONUCLEASE EndoG INDUCES ALTERNATIVE SPLICING 97
Fig. 4. Induction of splice variants of mRNA of TERT, Casp-2, DNase I, and BCL-x after EndoG overexpression. The expression levels of splice variants of mRNA of TERT, Casp-2, DNase I, and BCL-x in CD4+-Т lymphocytes of human (a–d), mouse (e–h), and rat (i–l), transfected with EndoG gene or incubated with cisplatin were shown. Gene expression levels were measured by real time RT-PCR and normalized using the expression level of the 18S rRNA reference gene. N = 4. *p ≤ 0.05 when compared with control cells; #p ≤ 0.05 when compared with cells transfected with pGFP.
98
ZHDANOV et al. (a)
(b)
Human TERT (127 kDa)
Casp-2 (51 kDa)
DNase I (31 kDa) L S L S L S L S
L S L S L S L S
(α+β+) pEndoG-GFP (α+β–) (α+β+) pGFP (α+β–) (α+β+) Cisplatin (α+β–) (α+β+) Control (α+β–)
Mouse TERT (127 kDa)
GAPDH (36 kDa)
BCL-x (26 kDa)
Casp-2 (51 kDa)
DNase I (31 kDa)
(α+β+) Cisplatin (α+β–) (α+β+) Control (α+β–)
BCL-x (26 kDa)
GAPDH (36 kDa)
L S L S L S L S
L S L S L S L S
(α+β+) pEndoG-GFP (α+β–) (α+β+) pGFP (α+β–)
(d)
(c)
(e) (α+β−)-TERT (α+β)-TERT 1.4 *# 1.0 TERT Casp-2 DNase I BCL-x GAPDH 0.9 *# 1.2 * *# (127 kDa) (51 kDa) (31 kDa) (26 kDa) (36 kDa) 0.8 * 1.0 L L * 0.7 (α+β+) pEndoG-GFP (α+β–) S S 0.6 0.8 *# * L L (α+β+) 0.5 pGFP (α+β–) 0.6 S S 0.4 *# * *# * L L 0.4 0.3 (α+β+) Cisplatin (α+β–) S S 0.2 0.2 L L (α+β+) 0.1 Control (α+β–) S S 0 0 Human Rat Human Rat Mouse Mouse pEndoG-GFP pGFP pEndoG-GFP pGFP Cisplatin Control Cisplatin Control (h) (f) (j) (i) (g) DNase I BCL-xL BCL-xS Casp-2L Casp-2S 0.9 1.0 1.2 1.6 1.4 *# *# *# 0.9 0.8 *# 1.4 * 1.2 1.0 *# * * 0.8 0.7 * 1.2 0.7 1.0 *# 0.6 0.8 * * 1.0 * 0.6 0.5 0.8 0.5 0.6 0.8 * * * 0.4 *# * *# * 0.4 0.6 *# 0.6 *# *# * * 0.3 * 0.4 0.3 *# *# *# *# 0.4 0.4 0.2 0.2 0.2 0.2 0.1 0.1 0.2 0 0 0 0 0 Human Rat Human Rat Human Rat Human Rat Human Rat Mouse Mouse Mouse Mouse Mouse pEndoG-GFP pGFP pEndoG-GFP pGFP pEndoG-GFP pGFP pEndoG-GFP pGFP pEndoG-GFP pGFP Cisplatin Control Cisplatin Control Cisplatin Control Cisplatin Control Cisplatin Control (l) (k) Human Mouse Rat Expression relatively to GAPDH
Expression relatively to GAPDH
Telomerase 100 90 80 70 60 50 *# * 40 *# * * 30 20 10 0 Human Rat Mouse pEndoG-GFP pGFP Cisplatin Control
Activity, %
pE
nd o p G GFP GF P C isp la tin C on tro l
Expression relatively to GAPDH
nd o p G GFP GF P C isp la tin C on tro l
pE
pE
nd o p G GFP GF P C isp la tin C on tro l
Expression relatively to GAPDH
Expression relatively to GAPDH
Expression relatively to GAPDH
Expression relatively to GAPDH
Rat
Rat
(o)
Absorption, conventional units
pG
nd pE
Absorption А505, conventional units
M
Mouse
oG FP GF P C isp la C tin on tro pE l nd o p G GF GF C P P isp C latin on tro pE l nd o p G GFP GF P C isp C latin on tro l
Human
(m) (n) Casp-2 0.35 0.30 kDa 0.25 150 * 0.20 100 *# * 0.15 *# 75 0.10 *# * 37 0.05 25 0 Human Rat 20 Mouse 10 pEndoG-GFP pGFP Cisplatin Control
DNase I 90 80 70 60 50 * 40 * *# * *# 30 *# 20 10 0 Human Rat Mouse pEndoG-GFP pGFP Cisplatin Control
Fig. 5. An inhibition of enzyme activity of TERT, Casp-2, DNase I, and BCL-x proteins after the induction of an alternative splicing of coding mRNA in human, murine, and rat cells transfected with EndoG gene or incubated with cisplatin. Western blotting of TERT, Casp-2, DNase I, BCL-x, and reference protein GAPDH in human, murine, and rat CD4+-Т lymphocytes (a, b, and c, respectively) transfected with EndoG gene or incubated with cisplatin, and relative expression level of splice variants of mRNA of TERT, Casp-2, DNase I, and BCL-x (d–j) relatively to GAPDH were shown. Telomerase activity measured by TRAP method followed by electrophoresis in 12% polyacrylamide gel (k) and diagram of telomerase activity measured by this method (l) were shown. Diagram of enzyme activity of Casp-2 (m). DNA-SDS-PAGE zymogram of human, murine, and rat cells transfected with EndoG gene or incubated with cisplatin (n) and activity of DNase I measured by zymography (o) were shown. N = 4. *p ≤ 0.05 relatively to control cells; #p ≤ 0.05 relatively to cells transfected with pGFP. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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APOPTOTIC ENDONUCLEASE EndoG INDUCES ALTERNATIVE SPLICING
Cisplatin
Control 100
80
41.80%
80
60
60
40
40
20
20
3.08%
0 0 1e0 1e–1 1e1 1e2 1e3 1e0 1e–1 1e1 1e2 1e3
(c)
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(b) 80
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65.44%
60
60
40
40
20
20
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Mouse
(a)
4.69%
0 0 1e0 1e–1 1e1 1e2 1e3 1e0 1e–1 1e1 1e2 1e3
80 70 60 50 40 30 20 10 0
99
* *
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Cisplatin Mouse EndoG GAPDH (33 kDa) (36 kDa)
(e) 1.4
*
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*
1.0 0.8
Cisplatin Control
0.6
(f)
0.4 0.2 0 Mouse Cisplatin
Rat Control
Rat EndoG GAPDH (33 kDa) (36 kDa)
Control
(g) 1.2 Expression relatively to GAPDH
Normalized expression level, conventional units
(d)
Rat
TUNEL
*
1.0
*
0.8 0.6 0.4 0.2 0
Cisplatin
Mouse Cisplatin
Control
Rat Control
Fig. 6. Induction of DNA damage and EndoG expression in CD4+-T lymphocytes of mice and rats 72 hours after intravenous injection of cisplatin at a dose of 60 mg/kg. Number of lymphocytes of mouse (a) and rat (b) with damaged DNA measured by TUNEL for flow cytometry, and an increase of the number of TUNEL-positive cell after cisplatin injection (c) were shown. EndoG expression measured by real-time RT-PCR in CD4+-Т cells of mice and rats after cisplatin injections (d); Western blotting of EndoG and reference protein GAPDH in cells of mice (e) and rats (f), and diagram of the ration of EndoG amount to the GAPDH amount (g) were shown. N = 4. *p ≤ 0.05 relatively to the cells of control animals.
28]. The full-length version of BCL-xL inhibits the function of the p53 protein and has an antiapoptotic effect [29]. Deletion of 189 nucleotides in the mRNA molecule causes the synthesis of the truncated variant BCL-xS that is a proapoptotic protein. DNA damage caused by the action of antitumor drugs leads to an increase in the level of BCL-xS mRNA and the activation of apoptosis in vitro [30, 31], which is consistent with the cisplatin activity shown by us and the activation of EndoG. To confirm the results of RNA sequencing, we examined the expression levels of EndoG and splice variants of TERT, Casp-2, DNase I, and BCL-x in intact CD4+-T cells of 12 healthy donors, as well as mice and rats of different lines (Fig. 1). Correlation analysis showed that increased expression of EndoG is accompanied by increased expression of truncated splice variants of mRNA of these genes (Fig. 2). Realtime RT-PCR and Western blotting confirmed the change in the number of splice variants of mRNA and proteins of the studied genes in CD4+-T cells transfected with plasmid pEndoG-GFP or incubated with cisplatin (Figs. 3 and 4). When the ratio of the splice RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
variants was changed, the enzymatic activity of the studied proteins decreased in the cells of the humans, mice and rats (Fig. 5). Cisplatin-induced DNA damage in mice and rats was accompanied by an increase in AS of TERT, Casp-2, DNase I, and BCL-x and a decrease in their enzymatic activity in vivo (Figs. 5 and 7). In the present study, we showed that the apoptotic endonuclease EndoG is capable of inducing the AS of mRNA of the catalytic subunit of telomerase TERT, Casp-2, DNase I, and BCL-x in human, murine and rat CD4+-T cells. Induction of AS resulted in inhibition of the enzymatic activity of these proteins in vitro and in vivo. The results obtained indicate the involvement of EndoG in the regulation of the enzymatic activity of various proteins and, consequently, in the determination of the cell fate in various organisms. EXPERIMENTAL Laboratory animals. Work with laboratory animals was approved by the ethical committee of the Orekhovich Institute of Biomedical Chemistry. In the experiments following, murine and rat lines were used: Vol. 44
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1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
(g)
*
*
TERT (127 kDa)
(k)
Mouse
Rat Mouse (α+β+)-TERT (α+β–)-TERT
СisplatinСontrolСisplatinСontrol
*
α+β+ α+β–
Control
TERT *
α+β+ α+β–
Cisplatin
(e)
Mouse Rat (α+β+)-TERT (α+β–)-TERT
L S L S
СisplatinСontrolСisplatinСontrol
*
*
TERT *
(b)
Rat
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
(h)
*
* *
Casp-2 *
*
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L S L S
*
BCL-x (26 kDa)
GAPDH (36 kDa)
Rat
100 90 80 70 60 50 40 30 20 10 0
(l)
Mouse
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*
Rat
*
Telomerase
Casp-2L Casp-2S
Rat
*
Mouse Rat
*
*
L S L S
*
DNase I
TERT (127 kDa)
Casp-2 (51 kDa)
Mouse
25 20 10
75 37
kDa M 150 100
(n)
Rat
Rat
*
*
*
*
BCL-x *
*
BCL-x
GAPDH (36 kDa)
*
Mouse
*
Rat
*
DNase I
СisplatinСontrolСisplatinСontrol Mouse Rat BCL-xL BCL-xS
*
*
BCL-x (26 kDa)
СisplatinСontrolСisplatinСontrol Rat Mouse BCL-xL BCL-xS
70 60 50 40 30 20 10 0
(o)
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
(j)
L S L S
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
(d)
DNase I (34 kDa)
СisplatinСontrolСisplatinСontrol Mouse Rat DNase I Δ4DNase I
Casp-2
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 *
Control α+β+ α+β–
(i)
*
DNase I
СisplatinСontrolСisplatinСontrol Rat Mouse DNase I Δ4DNase I
*
*
*
Cisplatin α+β+ α+β–
(f)
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
(c)
0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0
(m)
СisplatinСontrolСisplatinСontrol
*
*
*
DNase I (34 kDa)
Mouse
Mouse Casp-2L Casp-2S
СisplatinСontrolСisplatinСontrol
Casp-2 (51 kDa)
0
0.05
0.10
0.15
0.20
0.25
С i sp lat in Сo nt ro l С i sp lat in Сo nt ro l
Normalized expression level ×10е–3, conventional units
Expression relatively to GAPDH
Normalized expression level, conventional units Expression relatively to GAPDH
10 9 8 7 6 5 4 3 2 1 0
A505, a.u.
(a)
Activity, %
Normalized expression level, conventional units Expression relatively to GAPDH
Normalized expression level, conventional units Expression relatively to GAPDH
С i sp Сo latin n Сi trol sp l С atin o nt ro l
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Absorbance, a.u.
100 ZHDANOV et al.
Fig. 7. Induction of splice variants and inhibition of the enzymatic activity of the studied proteins 72 hours after intravenous injection of cisplatin at a dose of 60 mg/kg. Diagrams of expression levels of splice variants of TERT (a), Casp-2 (b), DNase I (c), and BCL-x (d) mRNA in human, murine, and rat CD4+-T lymphocytes; Western blotting results of TERT, Casp-2, DNase I, BCL-x, and the reference protein GAPDH in CD4+-T lymphocytes of mice (e) and rats (f); diagrams of the number of splice variants of the proteins relatively to the amount of GAPDH (g–j) were shown. Results of analysis of telomerase activity in murine and rat cells by TRAP method followed by electrophoresis in 12% acrylamide gel (k) and diagram of telomerase activity measured by this method (l). Diagram of Casp-2 enzyme activity (m) was shown. DNA-SDS-PAGE zymogram of murine and rat cells (n) and diagram of DNase I activity in murine and rat cells measured by zymography method (o). N = 4. *p ≤ 0.05 relatively to the cells of control animals.
2018
APOPTOTIC ENDONUCLEASE EndoG INDUCES ALTERNATIVE SPLICING
outbred mice of the ICR (CD-1) line and inbred mice of the BALB/c, C57BL/10, C57BL/6, DBA/2, SJL, C3H and CBA lines aged 6–9 weeks, as well as outbred SD (Sprague Dawley) and Wistar and inbred rats of WKY (Wistar-Kyoto), SHR and DA (Dark Agouti) lines at the age of 8–11 weeks. All animals came from the Pushchino breeding center for laboratory animals. Mice and rats were kept in a vivarium in the absence of a pathogenic microflora under natural light, on briquetted food and constant access to water. Blood sampling, selection and cultivation of CD4+ T cells. Samples of venous blood from four healthy individuals who gave written consent to participate in this study were taken into tubes with an anticoagulant K3EDTA (Greiner Bio-One, Austria). Blood mononuclear cells were isolated by gradient centrifugation at Ficole Lympholite-H (Cedarlane, Canada). The preparation of the CD4+ cell fraction was performed by magnetic selection using the human CD4+ Isolation Kit (Miltenyi Biotec, Germany) according to the manufacturer’s protocol. The resulting CD4+ cells were seeded at a concentration of 5 × 105 cells per mL of medium. The culture medium RPMI-1640 (Life technologies, United States) containing 10% FBS (Fetal Bovine Serum, Gibco, United States), growth factors IL-2 (100 U/mL, R&D Systems, United States), anti-CD3 antibodies (5 μg/mL, MedBioSpectrum, Russia), anti-CD28 antibodies (2 μg/mL, eBiosciences, United States), as well as penicillin (50 U/mL) and streptomycin (50 mg/mL) (both Sigma, United States) [12]. Selection of CD4+ T cells from C57BL/6 mice and Wistar rats was performed using a murine CD4+ T Cell Isolation Kit (Miltenyi Biotec). CD4+ cells of mice and rats were cultured in the medium described above with addition of 1 mM sodium pyruvate (Applichem, Germany), 10 mM HEPES (Sigma), and 0.02 mM 2-mercaptoethanol (Sigma). Cell cultivation was carried out in a CO2 incubator at 37°C, 5% CO2 and 90% humidity. After every three days of cultivation, the cells were seeded to a concentration of 5 × 105 cells per mL of medium. Counting the number of stained with trypan blue cells was performed on the Vi-cell XR Viability Cell Analyzer (Beckman Coulter, United States). Photography of the proliferating cells was carried out using an inverted Microscope Leica DMI300 (Leica Biosystems, United States). For in vivo experiments, C57BL/6 mice or Wistar rats were intravenously injected with cisplatin at a concentration of 20 mg/kg [25]. After 72 hours, the animals were euthanized in a CO2 chamber, blood was taken by puncture of the heart and CD4+-T lymphocyte selection was performed as described above. Cell treatment with cisplatin and transfection. To induce DNA damage and EndoG expression, CD4+-T cells were cultivated with cisplatin (cis-diamine RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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dichloroplatinum (II), Sigma) at a concentration of 60 μM for 96 hours [11]. Transfection of human, murine and rat CD4+-T cells was performed by one of the following plasmids: Human pEndoG-GFP plasmids, Mouse pEndoGGFP, Rat pEndoG-GFP, respectively, or the control plasmid pGFP. Plasmids based on the vector pGFP-N1 were synthesized by Clontech (United States). Transfection was performed using Lipofectamine 2000 (Invitrogen, United States) according to the manufacturer’s protocol. Transfection efficiency (usually 90– 100%) was estimated by fluorescence microscopy when counting GFP-positive cells. RNA sequencing. From the transfected, cisplatinincubated or control cells, total RNA was isolated and cDNA libraries were obtained with Illumina TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat (Illumina Inc., United States) [32, 33]. Each cDNA sample was diluted to a concentration of 6 pM and sequenced with HiScanSQ (Illumina Inc.) in the Total RNA-Seq application as described in [34]. The sequencing results were analyzed for the UCSC Homo sapiens reference genome hg19, Mus musculus reference genome mm10 and Rattus norvegicus reference genome rn10 using TopHat v1.3.0 [35]. Extraction of RNA and RT-PCR in real time. The total RNA from the cells was isolated using the RNeasy Mini Kit (Qiagen, United States) according to the manufacturer’s protocol. Reverse transcription and real-time PCR were performed according to the procedure described earlier [26]. For each probe, 5 μg of total RNA was subjected to a reverse transcription reaction in 25 μL of the reaction mixture (Invitrogen, United States) according to the manufacturer’s protocol. Platinum SYBR Green qPCR Supermix-UDG (Invitrogen, United States) was used as the reaction mixture for real-time PCR under the manufacturer’s protocol. Primers were synthesized by Synthol (Russia). For amplification, a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, United States) and a two-temperature mode (primer annealing/elongation) were used. The number of amplifications was determined by fluorescence at the end of the elongation cycle. Standard PCR efficiency curves were constructed from serial dilutions (1 : 40, 1 : 80, 1 : 160, and 1 : 320) of total cDNAs. Data were presented in the form of normalization of mRNA levels of the studied genes for the gene coding 18S rRNA (reference gene with constitutive expression). Western blotting. The cells were disrupted in 1 mL TBE buffer (89 mM Tris, 89 mM H3BO3, 2 mM EDTA, pH 8.0) by sonication for 2 min at 50 W capacity with a Model 50 Sonic Dismembrator homogenizer (Fisher Scientific, United States) and centrifuged for 10 min at 12000g to remove the debris. The total protein concentration in the samples was measured with Bradford Protein Assay (Pierce, United Vol. 44
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States) using bovine serum albumin to construct a calibration curve. The cell lysate (based on 50 μg of total protein) was dissolved in 50 mM Tris-HCl, pH 6.8 containing 1% SDS, 2 mM EDTA, 1% 2-mercaptoethanol and 7.5% glycerol, denatured by heating at 100°C for 10 min and separated by gradient PAGE [36] (100 V, 2 h) using NuPAGE® Novex® 4-12% Bis-Tris Protein Gels (Life Technologies, United States). The proteins were then transferred to a nitrocellulose membrane in Novex Transferring Buffer (Invitrogen, United States) at 40 V for 3 h, after which the membranes were stained with Ponseau S (Sigma, United States) as described by Hofnagel et al. [37]. The membranes were then blocked with Blotting-Grade Blocker (Bio-Rad, United States) and incubated for 2 hours with primary antibodies: polyclonal antiEndoG antibodies (Millipore, United States) diluted 1 : 500; monoclonal anti-GAPDH diluted 1 : 1000; anti-hTERT diluted 1 : 1000; anti-Casp-2 diluted 1 : 1000; antihuman BCL-x diluted 1 : 1000 (all from Abcam, United States) or antimouse, antirat DNase I diluted 1 : 500 (Rockland, United States). Then, the primary antibodies were washed out in phosphate buffer saline (PBS), pH 7.6, with 0.1% Tween-20, and the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (Cell Signalling, United States). SuperSignal Сhemiluminescent Kit (Pierce Biotechnology, United States) was used for visualization with the subsequent documentation with ChemiDoc™ XRS Imaging System (Bio-Rad, United States). A quantitative evaluation of the protein content was performed by the densitometry method with the GelAnalyzer 2010a program. Estimation of telomerase, Casp-2, and DNase I activities. The activity of telomerase was determined in cell lysates by the TRAP method (Telomeric Repeat Amplification Protocol), as described previously [5, 6]. Determination of the enzymatic activity of Casp-2 in cell lysates was carried out in a 96-well black plastic plate (Corning, United States) using the Caspase 2 Assay Kit (Fluorometric, Abcam) according to the manufacturer’s protocol. Fluorescence was measured in a MultiscanGo (Thermo Scientific) plate spectrometer at excitation and emission wavelengths of 400 and 505 nm, respectively. The DNase I enzymatic activity was determined by the method of DNA-SDS-PAGE zymography, described by Basnakian et al. [38]. The cells were homogenized in a buffer containing 0.25 M sucrose, 50 mM Tris-HCl pH 7.4 and 5 mM β-mercaptoethanol. A solution of 60 μg protein in 30 μL buffer containing 50 mM Tris-HCl, pH 6.8, 2 mM EDTA, 1% 2-mercaptoethanol, 7.5% glycerol, and 0.01% bromophenol blue was applied to 10% PAGE containing 0.1% SDS and calf thymus denatured DNA (Sigma). Electrophoresis was performed at a constant amperage of 35 mA for 1 h in a concentrating gel and at 75 mA for 3 h in a separating gel. After electrophoresis, the gel
was washed in 150 mL of milk (7.5 g, Bio-Rad) for 1 h at room temperature and incubated overnight in 150 mL of milk containing 40 mM mM Tris-HCl pH 7.4, 5 mM MgCl2, 2 mM CaCl2, and 0.02% sodium azide for activation of DNase I. The visualization of the gel was carried out by staining in a 1% solution of ethidium bromide for 30 min. The zones of DNase I activity were uncolored in UV light. Statistical analysis. Statistical analysis of the results was carried out by the Student’s test using Statistica 9.0 software (StatSoft Inc., United States). Results were represented as mean ± standard deviation. Statistical significance was achieved when p ≤ 0.05. To study the relationship between the level of expression of EndoG and the number of splice variants of the proteins studied, the cells ranked by the level of expression of EndoG, were divided into two groups: cells with an expression level of EndoG below the median were assigned to a group of cells with low expression of apoptotic endonuclease, with an expression of EndoG above the median, to a group of cells with high expression of the enzyme. In these groups, the Pearson correlation analysis of EndoG expression levels and hTERT splice variants was performed using the Statistica 9.0 software. ACKNOWLEDGMENTS The work was carried out with the financial support of the Program of Fundamental Scientific Research of the State Academies of Sciences for 2013-2020, as well as scholarships of the President of the Russian Federation for studying abroad for D.D. Zhdanov. The authors are grateful to Professor A.G. Basnakian (University of Arkansas for Medical Sciences, Little Rock, AR, United States) for help with the RNA sequencing. REFERENCES 1. Kim, E., Magen, A., and Ast, G., Nucleic Acids Res., 2007, vol. 35, p. 125. 2. Makeyev, E.V., Zhang, J., Carrasco, M.A., and Maniatis, T., Mol. Cell, 2007, vol. 27, pp. 435–448. 3. Jin, Y., Yang, Y., and Zhang, P., RNA Biol., vol. 8, pp. 450–457. 4. Chen, M. and Manley, J.L., Nat. Rev. Mol. Cell Biol., 2009, vol. 10, p. 741. 5. Zhdanov, D.D., Vasina, D.A., Orlova, E.V., Orlova, V.S., Pokrovskaya, M.V., Aleksandrova, S.S., and Sokolov, N.N., Biomed. Khim., 2016, vol. 62, pp. 544–554. 6. Zhdanov, D.D., Vasina, D.A., Orlova, V.S., Gotovtseva, V.Y., Bibikova, M.V., Pokrovsky, V.S., Pokrovskaya, M.V., Aleksandrova, S.S., and Sokolov, N.N., Biomed. Khim., 2016, vol. 62, pp. 239–250. 7. Khanna, A. and Stamm, S., RNA Biol., 2016, vol. 7, pp. 480–485. 8. Bauman, J., Jearawiriyapaisarn, N., and Kole, R., Oligonucleotides, 2009, vol. 19, pp. 1–13.
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